The present invention discloses probiotic Lactobacillus casei preparations. Specifically, anti-infective, anti-diarrhea preparations derived from a newly characterized strain of Lactobacillus casei designated KE01 are disclosed. Related methods for preparing the probiotic compounds using Lactobacillus casei strain KE01 and related methods for using the KE01 probiotic compositions are also disclosed.
The newly recognized probiotic Lactobacillus casei strain described herein has been designated KE01. Previously, this same organism had been designated Lactobacillus casei KE99 (see for example U.S. provisional application No. 60/256,528 and A. S. Naidu, X. Xie, D. A. Leumer, S. Harrison, M. J. Burrill and E. A. Fonda. 2001. Reduction of Sulfide, Ammonia Compounds and Adhesion Properties of Lactobacillus casei strain KE99 In Vitro. Curr. Microbiol. 43: In press.)
Lactic acid bacteria (LAB) are indigenous microflora of mammalian gastrointestinal tract that play an important role in the host microecology and have been credited with an impressive list of therapeutic and prophylactic properties. These therapeutic and prophylactic properties include, but not limited to the maintenance of microbial ecology of the gut, physiological, immuno-modulatory and antimicrobial effects. Other LAB associated attributes include enzyme release into the intestinal lumen that act synergistically with LAB adhesion to alleviate symptoms of intestinal malabsorption. Furthermore, the LAB enzymes help regulate intestinal pH which results in increased aromatic amino acid degradation. [Fuller, R. Probiotic foodsxe2x80x94current use and future developments. IFI NR 3:23-26 (1993); Mitsuoka, T. Taxonomy and ecology of Bifidobacteria. Bifidobacteria Microflora 3:11 (1984); Gibson, G. R. et al., Probiotics and intestinal infections, p.10-39. In R. Fuller (ed.), Probiotics 2: Applications and practical aspects. Chapman and Hall, London, U.K. (1997); Naidu A S, et al., Probiotic spectra of lactic acid bacteria (LAB). Crit Rev Food Sci Nutr 39:3-126 (1999); Naidu, A. S., Clemens, R. A. Probiotics, p.431-462. In A. S. Naidu (ed.), Natural Food Antimicrobial Systems. CRC Press, Boca Raton, Fla. (2000)]
Lactic acid bacteria have also demonstrated the ability to significantly reduce sulfide and ammonia containing compounds in animal fecal waste and thus reduce the odor and toxicity associated with animal excrements. This ex vivo LAB application is becoming increasingly more important as agro-businesses expand and as communities continue their seemingly never ending encroachment into previously unoccupied rural areas. For example, LAB has been demonstrated to eliminate offensive odors and reduce hydrogen sulfide production associated with hatchery waste when cockerel chicks and shell waste are blended with a mixture containing 15% carbohydrate and LAB. Moreover, LAB compositions have demonstrated efficacy in diminishing the Escherichia coli and Salmonella content of hatchery waste to negligible levels. Additionally, the odor and viscosity of poultry offals such as broiler-processing waste is significantly reduced by L. acidophilus mediated lactic acid fermentation. Furthermore, preparations containing LAB have been reported to accelerate the breakdown of hard-to-degrade carbohydrates and decrease the ammonia production by porcine cecal bacteria. Finally, ex vivo L. casei FG1 and L. plantarum FG10 silage fermentation significantly reduces ammonia levels by inhibiting urea-splitting organisms. [Deshmukh, A. C., Patterson, P. H. Preservation of hatchery wastes by lactic acid fermentation. 1. Laboratory scale fermentation. Poult Sci 76:1212-1219 (1997); Russell, S. M. et al., Lactic acid fermentation of broiler processing waste: physical properties and chemical analyses. Poult Sci 71:765-770 (1992); Tibbetts, G. W. et al., Poultry offal ensiled with Lactobacillus acidophilus for growing and finishing swine diets. J Anim Sci 64:182-190 (1987); Sakata, T. et al., Probiotic preparations dose-dependently increase net production rates of organic acids and decrease that of ammonia by pig cecal bacteria in batch culture. Dig Dis Sci 44:1485-1493 (1999); Cai, Y. et al., Effect of applying lactic acid bacteria isolated from forage crops on fermentation characteristics, aerobic deterioration of silage. J Dairy Sci 82:520-526 (1999); Modler, H. W. et al., Bifidobacteria and bifidogenic factors. Can Inst Food Sci Tech 23:29-41 (1990)].
However, the greatest potential for LAB to improve life quality for man and domestic animals lies in LAB in vivo probiotic applications. In order for LAB to exhibit beneficial probiotic effects in vivo, the organisms must survive for extended time periods in the gastrointestinal tract. Therefore, it is critical that probiotic LAB strains be selected that possess qualities that prevent their rapid removal by gut contraction. Effective probiotic bacteria must able to survive gastric conditions and colonize the intestine, at least temporarily, by adhering to the intestinal epithelium. Consequently, LAB that demonstrate an enhanced ability to adhere to mucosal surfaces, and therefore possess improved bacterial maintenance and prolonged gastrointestinal tract residence times, have a competitive advantage over LAB that do not. [Salminen, S. et al., Clinical uses of probiotics for stabilizing the gut mucosal barrier: successful strains and future challenges. Antonie Van Leeuwenhoek 70:347-358 (1996); Conway, P. Selection criteria for probiotic microorganisms. Asia Pacific J Clin Nutr 5:10-14 (1996); Havenaar, R. et al., Selection of strains for probiotic use, p.209-224. In R. Fuller (ed.), Probiotics, the scientific basis. Chapman and Hall, London, U.K. (1992)].
Lactobacillus can successfully colonize the mammalian gastrointestinal tract through a number of different mechanisms. For example, some bacterial species bind to various sub-epithelial matrix proteins and specific receptors on the intestinal mucosa. Other species may adhere to mammalian intestinal cells via mechanisms that involve different combinations of carbohydrate and protein factors on the bacteria and host eucaryotic cell surfaces. However, regardless of the mechanism(s) of attachment, it is the ability of LAB to successfully colonize the human gastrointestinal tract that provides LAB with probiotic qualities. [Greene, J. D., Klaenhammer, T. R. Factors involved in adherence of lactobacilli to human Caco-2 cells. Appl Environ Microbiol 60:4487-4494 (1994); Sarem, F. et al., Comparison of the adherence of three Lactobacillus strains to Caco-2 and Int-407 human intestinal cell lines. Lett Appl Microbiol 22:439-442 (1996); Naidu, A. S., et al., Particle agglutination assays for rapid detection of fibronectin, fibriogen, and collagen receptors on Staphylococcus aureus. J Clin Microbiol 26:1549-1554 (1988); Wadstrom, T. et al., Surface properties of lactobacilli isolated from the small intestines of pigs. J Appl Bacteriol 62:513-520 (1987); Bernet, M. F. et al., Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment, invasion by entero-virulent bacteria. Gut 35:483-489 (1994); Jin, L. Z. et al., Effect of adherent Lactobacillus spp. on in vitro adherence of salmonellae to the intestinal epithelial cells of chicken. J Appl Bacteriol 81:201-206 (1996); Reid, G. et al., Influence of lactobacilli on the adhesion of Staphylococcus aureus and Candida albicans to fibers and epithelial cells. J Ind Microbiol 15:248-253 (1995)].
Generally speaking probiotic bacteria exert their beneficial effects by displacing invasive or toxigenic pathogenic enteric bacteria (enteric pathogens) from the intestinal mucosa through a competitive binding process. Enteric pathogens such as, but not limited to enteropathogenic Escherichia coli (EPEC), enterotoxigeneic E. coli (ETEC), Salmonella enteriditis, Yersina pseudotuberculosis and Listeria monocytogenes must be able to successively colonize an animal""s intestinal tract in order to cause disease.
The mechanisms these organisms use to effectively colonize the intestine are varied. For example, ETEC bearing CFA/I or CFA/II adhesive factors specifically adhere to the brush border of the polarized epithelial human intestinal Caco-2 cells in culture. S. typhimurium and EPEC adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Y. pseudotuberculosis and L. monocytogenes bind to the periphery of undifferentiated Caco-2 cells.
Recently, heat-killed L. acidophilus preparations have been proven to be effective probiotic compositions. Heat-killed L. acidophilus preparations have been shown to displace known enteric pathogens from the lining of a test animal""s intestinal wall in a dose dependent manner. Consequently, enteric pathogens were unable to colonize the animal""s gastrointestinal tract thus preventing disease. For example, L. acidophilus (Lacteol(copyright) strain) was found to inhibit this adhesion in a dose-dependent manner of E. coli strain B41 (ECB41). In other experiments live and heat-killed L. acidophilus strain LB successfully inhibited both Caco-2 cell association and invasion of enteric pathogens. An in yet another study, heat-killed L. acidophilus strain LB was shown to completely inhibit ETEC adhesion to Caco-2 cells. [Fourniat, J. et al., Heat-killed Lactobacillus acidophilus inhibits adhesion of Escherichia coli B41 to HeLa cells. Ann Rech Vet 23:361-370 (1992); Chauviere, G. et al., Competitive exclusion of diarrheagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 cells by heat-killed Lactobacillus. FEMS Microbiol Lett 70:213-217 (1992)]. Coconnier, M. H. et al., Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion. FEMS Microbiol Lett 110:299-305 (1993)].
As previously explained, probiotic compositions are generally defined as microbial dietary supplements that beneficially affect the host by improving intestinal microbial balance. The two major genera of microorganisms commonly associated with probiotics include Lactobacillus sp and Bifidobacteria sp. The beneficial effects attributed to probiotics include increased resistance to infectious diseases, healthier immune systems, reduction in irritable bowel syndrome, reductions in blood pressure, reduced serum cholesterol, milder allergies and tumor regression. However, in spite of recent scientific advances and the publication of limited in vivo and in vitro experimental evidence supporting the efficacy of probiotic compositions, the major studies reporting these and other benefits have relied on antidotal evidence.
Examples of publications depending on antidotal and limited in vitro data include U.S. Pat. Nos. 3,957,974, 4,210,672, 4,314,995, 4,345,032, 4,579,734, 4,871,539, 4,879,238 and 5,292,362 (the xe2x80x9cHataxe2x80x9d patents). The Hata patents disclose various subspecies of Lactobacillus spp and report beneficial probiotic-like effects. However, the Hata patents do not disclose or describe a Lactobacillus species that demonstrates avid binding to sub-epithelial matrices and competitive exclusion and microbial interference with bacterial enteric pathogens.
U.S. Pat. No. 6,060,050 (the xe2x80x9c""050 patentxe2x80x9d) discloses a preparation consisting of probiotic microorganisms dispensed in a starch containing medium used to protect the organism during storage, serves to transport the organism to the large bowel and provides a growth promoting substrate. However, the ""050 patent does not provided data that directly demonstrates probiotic activity against enteric pathogens.
U.S. Pat. No. 5,965,128 (the xe2x80x9c""128 patentxe2x80x9d) discloses the treatment of enteric diseases caused by E. coli O157:H7 (enterohemorrhagic E. coli). However, the probiotic compositions of the ""128 patent only includes non-pathogenic strains of E. coli, no Lactobacillus sp are disclosed. Consequently, while the non-pathogen enteric organisms of the ""128 patent may afford the recipient xe2x80x9cprobiotic-likexe2x80x9d protection against enterohemorrhagic E. coli, other beneficial qualities associated with Lactobacillus sp and Bifidobacteria sp are absent.
U.S. Pat. Nos. 4,839,281, 5,032,399 and 5,709,857 (the xe2x80x9c""857 patentxe2x80x9d) discloses several strains of Lactobacillus acidophilus that adhere to intestinal mucosal cells. Moreover, the ""857 patent discloses a Lactobacillus acidophilus strain that inhibits the growth of certain enteric pathogens. However, the ""857 patent does not disclose L. casei strains having the proven beneficial qualities associated with the L. acidophilus cultures of the ""857 patent.
Therefore, there remains a need for new strains of Lactobacillus sp. that exhibit probiotic activities. Particularly, there is a need for more strains of Lactobacillus sp. that have the proven capacity to reduce enteric pathogen diseases and increase animal vitality and health.
It is an object of the present invention to provide a new strain of Lactobacillus sp that has demonstrated probiotic properties.
It is another object of the present invention to provide a new strain of Lactobacillus spp that has anti-enteric pathogen probiotic activity.
It is yet another object of the present invention to provide a new strain of Lactobacillus sp. that maintain animal health and vitality.
It is still another object of the present invention to provide dietary supplements and pharmaceutical preparations made from Lactobacillus sp. having demonstrated probiotic properties.
The present invention fulfills these and other objects by providing a new strain of Lactobacillus casei designated KE01 that possesses scientifically proven probiotic properties including demonstrated in vivo anti-enteric pathogen activity. Moreover, the present invention provides dietary supplements and pharmaceutical preparations composed of L. casei strain KE01 that are formulated in a sugar complex composed of trehalose and fructooligosaccharides that provides long term protection to the organism and helps maintain its proven probiotic properties.
There is a need for new probiotic formulations that can be used to treat and prevent enteric-pathogen infections and help maintain the health and vitality of humans and livestock. Recently, the Federal Food and Drug Administration (FDA) has intensified its campaign against the over prescription and clinical abuse of antibiotics. The excessive use of antibiotics has increased in the number of human and animal pathogens that are resistant to first-line antibiotics resulting in an increase in infections that do not respond to conventional antimicrobial therapies. Moreover, the prophylactic use of antibiotics in animal feed has resulted in an alarming increase in livestock intestinal infections resulting in diminished herd size and animal weight due to nutrient malabsorption. Consequently, the number of healthy animals suitable for human consumption has dropped, and those that do survive long enough to reach market have significantly lower weights and consequently reduced meat quality.
One means of preventing the rapid spread of drug resistant enteric pathogens in humans and livestock is to significantly reduce antibiotic use. However, the spread of communicable diseases including enteric infections is inevitable due to over crowding of farms and cities. Consequently, before prophylactic antibiotic use can be completely discontinued a suitable antimicrobial alternative must be available. Recent studies have indicated that the use of foodstuffs and dietary supplements containing specific strains of probiotic microorganisms can help prevent, and in many cases actually cure, enteric pathogen diseases. However, many of the probiotic formulas currently marketed rely on organisms including Lactobacillus spp and Bifidobacteria sp (and other genera) that have not been subjected to scientific scrutiny using approved methods for assessing probiotic efficacy. Consequently, too many of the xe2x80x9cprobioticxe2x80x9d formulas currently available lack proven in vivo anti-enteric pathogen activity. Moreover, many of the clinically effective probiotic formulations commercially available are not stable upon storage and therefore do not deliver effective amounts of viable probiotic bacteria to the user. The present inventor has tested many commercially available preparations and found microbial viability well below stated concentrations and in many cases the present inventor has found that these commercial preparations did not contain any viable bacteria.
The present inventor has developed methods for preparing and packaging a new strain of L. casei, designated KE01. This new strain of L. casei was originally from a traditional fermented yoghurt-like Asian dairy product by the present inventor. Subsequently, the present inventor characterized the isolate and the strain deposited with the American Type Culture Collection (ATCC, MD, USA)). Lactobacillus casei strain KE01 has been given the ATCC depository number PTA-3945. Moreover, the present inventor has developed preparations that maintain L. casei KE01 viability such that a clinically effective dose of viable probiotic microorganisms reaches the host.
The present invention provides a L. casei strain (KE01) that interferes with bacterial adherence (microbial interference) of enteric pathogens such as, but not limited to enteropathogenic and enterotoxigenic E. coli, Helicobacter pylori, Campylobacter jejuni, S. typhimurium, and S. enteritidis to a variety of mammalian cell types. Moreover, the Lactobacillus of the present invention can also competitively exclude (competitive exclusion) these, and other bacterial pathogens, from binding to many mammalian cells. The beneficial properties associated with the novel Lactobacillus strain of the present invention have resulted in improved probiotic dietary supplements that support general human and animal health. Moreover, the present invention can be used to provide prophylactics, therapeutics and palliatives (collectively referred to herein as xe2x80x9cprobioticsxe2x80x9d) for conditions such as, but not limited to, traveler""s diarrhea, gastrointestinal infections, hemolytic uremic syndrome, and gastric ulcers.
Additional novel features and qualities of this new L. casei strain KE01 include, but are not limited to, L. casei KE01""s ability to reduce sulfide concentrations by a factor exceeding 300 ppm within 48 hours when exposed to a growth medium containing approximately 2000 ppm of sulfides and the demonstration of avid binding to sub-epithelial matrices including Bio-coat(trademark) (Collagen type-I, Collagen type IV, laminin, and fibronectin), Matrigel(trademark) and Caco-2 cell monolayer. Most importantly, a reconstituted, freeze-dried preparation of the L. casei of the present invention has been shown to effectively detach collagen-adherent E. coli. 
The methods used to maintain the viability of the L. casei of the present invention and preserve its E. coli displacement qualities include, but are not limited the use of the sugar trehalose and moisture and packaging the final compositions in oxygen proof polymer-lined (e.g. Mylar(copyright)) foil pouches.
These and other beneficial probiotic properties of the new strain of Lactobacillus will be further evident by the following, non-limiting, detailed description of the present invention.